open access publication

Article, 2023

Formate overflow drives toxic folate trapping in MTHFD1 inhibited cancer cells

In: Nature Metabolism, ISSN 2522-5812, Volume 5, 4, Pages 642-659, 10.1038/s42255-023-00771-5

Contributors (34)

Green, Alanna Claire (0000-0002-0175-1485) [1] Marttila, Petra S K (0000-0002-0115-8067) [2] Kiweler, Nicole (0000-0001-7493-703X) [3] Chalkiadaki, Christina [2] Wiita, Elisée [2] Cookson, Victoria J (0000-0003-3089-4837) [1] Lesur, Antoine [3] Eiden, Kim [3] Bernardin, François (0000-0002-6329-5542) [3] Vallin, Karl S A [2] [4] Borhade, Sanjay R [2] [5] Long, Maeve [2] Ghahe, Elahe Kamali [6] Jiménez-Alonso, Julio José (0000-0002-2476-2507) [2] [7] Jemth, Ann-Sofie (0000-0002-7550-1833) [2] Loseva, Olga [2] Mortusewicz, Oliver (0000-0002-4290-4994) [2] Meyers, Marianne [8] Viry, Elodie [3] Johansson, Annika I (0000-0001-5000-1288) [9] Hodek, Ondřej (0000-0001-8307-9575) [10] Homan, Evert J (0000-0002-9057-1848) [2] Bonagas, Nadilly (0000-0001-7336-4065) [2] Ramos, Louise [1] Sandberg, Lars [11] Frödin, Morten (0000-0003-3691-9820) [6] Moussay, Etienne (0000-0002-0879-8067) [3] Slipicevic, Ana [2] [12] Letellier, Elisabeth (0000-0001-8242-9393) [8] Paggetti, Jérôme (0000-0001-9460-5876) [3] Sørensen, Claus Storgaard (0000-0001-6022-9710) [6] Helleday, Thomas (0000-0002-7384-092X) (Corresponding author) [1] [2] Henriksson, Martin (Corresponding author) [2] Meiser, Johannes (0000-0002-9093-6210) (Corresponding author) [3]


  1. [1] University of Sheffield
  2. [NORA names: United Kingdom; Europe, Non-EU; OECD]
  3. [2] Karolinska Institutet
  4. [NORA names: Sweden; Europe, EU; Nordic; OECD]
  5. [3] Luxembourg Institute of Health
  6. [NORA names: Luxembourg; Europe, EU; OECD]
  7. [4] RISE Research Institutes of Sweden
  8. [NORA names: Sweden; Europe, EU; Nordic; OECD]
  9. [5] RedGlead Discover, Lund, Sweden


Cancer cells fuel their increased need for nucleotide supply by upregulating one-carbon (1C) metabolism, including the enzymes methylenetetrahydrofolate dehydrogenase–cyclohydrolase 1 and 2 (MTHFD1 and MTHFD2). TH9619 is a potent inhibitor of dehydrogenase and cyclohydrolase activities in both MTHFD1 and MTHFD2, and selectively kills cancer cells. Here, we reveal that, in cells, TH9619 targets nuclear MTHFD2 but does not inhibit mitochondrial MTHFD2. Hence, overflow of formate from mitochondria continues in the presence of TH9619. TH9619 inhibits the activity of MTHFD1 occurring downstream of mitochondrial formate release, leading to the accumulation of 10-formyl-tetrahydrofolate, which we term a ‘folate trap’. This results in thymidylate depletion and death of MTHFD2-expressing cancer cells. This previously uncharacterized folate trapping mechanism is exacerbated by physiological hypoxanthine levels that block the de novo purine synthesis pathway, and additionally prevent 10-formyl-tetrahydrofolate consumption for purine synthesis. The folate trapping mechanism described here for TH9619 differs from other MTHFD1/2 inhibitors and antifolates. Thus, our findings uncover an approach to attack cancer and reveal a regulatory mechanism in 1C metabolism.


MTHFD1, MTHFD2, accumulation, activity, antifolates, approach, cancer, cancer cells, cells, consumption, cyclohydrolase activity, de novo purine synthesis pathway, death, dehydrogenase, depletion, downstream, enzyme, findings, formate, formate overflow, formate release, hypoxanthine levels, inhibitors, levels, mechanism, metabolism, mitochondria, need, nucleotide supply, one-carbon metabolism, overflow, pathway, potent inhibitor, presence, purine synthesis, purine synthesis pathway, regulatory mechanisms, release, supply, synthesis, synthesis pathway, thymidylate depletion, trapping, trapping mechanism, traps


  • Sheffield Teaching Hospitals NHS Foundation Trust
  • Deutsche Forschungsgemeinschaft
  • Swedish Research Council
  • Swedish Cancer Society
  • Barncancerfonden
  • Ministry of Economy, Industry and Competitiveness
  • Novo Nordisk Foundation
  • Novo Nordisk (Denmark)
  • Fonds National de la Recherche
  • Ragnar Söderberg Foundation